AGO1 requires interaction with GW182 to repress translation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein (AGO) and a GW182 family protein. In turn, GW182 proteins interact with PABP and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the translational repression, deadenylation and decay of miRNA targets (Huntzinger at al., 2013). Recent studies have indicated that miRNAs can also repress translation in a GW182-independent but AGO-dependent manner (Fukaya and Tomari, 2013). AGO1 and GW182-mediated repression was reported to occur by distinct mechanisms in Drosophila melanogaster (Dm) cells (Fukaya and Tomari, 2013). However, the contribution of these two alternative mechanisms to silencing has remained unclear. To address this question, we characterized the interaction of Dm AGO1 and GW182. Based on the recent structure of human AGO2 (Schirle and MacRae, 2012), we designed mutations that disrupt Dm AGO1 interaction with GW182. Functional assays in Dm cells indicate that Dm AGO1 requires interaction with GW182 to mediate translational repression and degradation of mRNA targets and does not possess independent repressive activity. We are combining cellular, biochemical and structural approaches to investigate how the GW182 proteins repress translation of miRNA targets